1. Field of the Invention
This invention relates to a gene encoding flagellin and an excretion vector having a gene encoding flagellin or a part of said gene.
2. Prior Art
A recent development of gene engineering made it easier that a gene encoding certain peptide is inserted into a vector and then the vector is introduced into bacteria such as Escherichia coli to give a large amount of the peptide. However, since a peptide expressed in bacteria is accumulated therein, growth of the bacteria is inhibited by the expressed peptide and production of the peptide is restricted by negative-feedback through excess production of the peptide. In order to recover the peptide, it is necessary to collect and destroy the bacteria, and to purify the peptide from the cell debris. It contains a lot of impurities, some of which are pathogenic to human beings, so that it is hard to recover the pure peptide therefrom.
Peptides composing cell membrane, secreted peptides and so on are synthesized as pro-peptides having signal sequence at their N-terminal, which is necessary for their passing through inner or outer membrane. The pro-peptide, from which the signal sequence is cut off by peptidase in the membrane in passing through the membrane, becomes a mature peptide to show its original activity and function.
So as to remove the difficulty in refining the target peptide accumulated in cells and improve productivity of the target peptide, it has been attempted to make cells secrete a target peptide outside by utilizing the natural secretion system of the cell as noted above, for example, secretion of .beta.-lactamase of Escherichia coli by a secretion vector carrying promoter and DNA encoding signal sequence of .alpha.-amylase of Bacillus subtilis (J. Biochem. 95, 87-93 (1984)), secretion of mouse IFN-.beta. by the same vector system as noted above (Gene 34, 1-8 (1985)) and secretion of human IFN-.alpha. by a secretion vector carrying a promoter and DNA encoding signal sequence of alkaline phosphatase of Escherichia coli (J. Biochem. 97, 1429-1436 (1985)).
Other than the secretion systems as mentioned above, an excretion system is a well known mechanism so that a peptide synthesized in a cell goes out of the cell, for example, flagellin composing flagella of bacteria is excreted outside by this excretion system. In the excretion system, differing from the secretion system, a peptide synthesized in a cell having no signal sequence is excreted out and presents its function without digestion by peptidase (J. Bacteriol. 159, 1056-1059 (1984)). The gene encoding flagellin of E. coli is called "hag gene", which has already been cloned in pBR322 and phage .lambda. (Abstracts of the 57th annual meeting of Japanese society of genetics, 63 (1985); J. Bacterial. 130, 736-745 (1977)). Though its partial DNA sequence was analyzed (J. Bacterial. 155, 74-81 (1983)), the whole DNA sequence has not yet been determined.
A secretion of peptide depends upon a type of a secretion vector, that is, a certain type of vector carrying a certain promoter and DNA sequence encoding signal peptide can be used in secretion of only a few kinds of peptides but not all kinds of peptides.
The hag gene encoding flagellin of E. coli has already been cloned and its partial sequence has been determined but its whole sequence has not yet been determined. It has not been suggested at all that an excretion system of flagellin can be utilized in production of a target peptide outside of a cell. Moreover, an excretion vector carrying the hag gene has not yet been constructed. The elucidation of the whole sequence of the hag gene makes it easier to construct such a vector.
Now, even if a target peptide is secreted in a broth by utilizing a secretion vector, it is fairly difficult to recover and refine the peptide from the broth. However, if a target peptide is excreted as a fused peptide with flagellin and composes flagella to become insoluble, it becomes easier to recover the peptide.